Isolated TWIK-1 potassium channel proteins

ABSTRACT

This invention relates to the cloning of a member of a new potassium channel named TWIK-1. More specifically, it relates to an isolated and purified nucleic acid molecule coding for a protein constituting a potassium channel exhibiting the proper-ties and structure of the TWIK-1 type channel, as well as the protein coded by this nucleic acid molecule. The invention also relates to the use of this nucleic acid molecule to transform cells, and the use of these cells expressing the potassium channels exhibiting the properties and structure of the TWIK-1 type channel for the screening of drugs.

This application is a division of U.S. patent application Ser. No.08/749,816, filed 15 Nov. 1996, now U.S. Pat. No. 6,013,470.

The present invention relates to a new family of potassium channels.More specifically, the invention relates to the cloning of a humanpotassium channel that constitutes the first member of a new functionaland structural group of potassium channels. The abundance of thischannel and its presence in a large number of tissues are such as toconfer on it a fundamental role in the transport of potassium in a largenumber of types of cells.

Potassium channels are ubiquitous in eukaryote and prokaryote cells.Their exceptional functional diversity make them ideal candidates for alarge number of biological processes in living cells (Rudy, B., 1988,Neurosciences, 25, 729–749; Hille, B., 1992, “Ionic Channels ofExcitable Membrane”, 2nd edition, Sinauer, Sunderland, Mass.). Inexcitable cells, the K⁺ channels define the form of the actionpotentials and the frequency of the electric activity, and play a majorrole in neuronal integration, muscle contraction or hormonal secretion.In nonexcitable cells, their expression appears to be correlated withspecific stages of the development of the cell (Barres, B. A. et al.,1990, Annu. Rev. Neurosci., 13, 441–474). In most cells, specific typesof K⁺ channels play a vital role in determining the electrical potentialof the membrane at rest by regulating the membrane permeability to K⁺ions. These channels exhibit the characteristic of being instantaneousand open in a large range of membrane potentials.

Recent cloning studies have resulted in the identification of a largenumber of subunits capable of forming potassium channels (Betz, H.,1990, Biochemistry, 29, 3591–3599; Pongs, O., 1992, Physiol. Rev., 72,S69–88; Salkoff, L. et al., 1992, Trends Neurosci., 15, 161–166; Jan, L.Y. and Y. N. Jan, 1994, Nature, 371, 199–122; Doupnik, C. A. et al.,1995, Curr. Opin. Neurobiol., 5, 268–277) which could be regulated byother types of subunits (Aldrich, R. W., 1994, Curr. Biol., 4, 839–840;Isom, L. L. et al., 1994, Neuron, 12, 1183–1194; Rettig, J. et al.,1994, Nature, 369, 289–294; Attali, B. et al., 1995, Proc. Natl. Acad.Sci. USA, 92, 6092–6096).

The subunits of the voltage-dependent K⁺ channels activated bydepolarization (Kv families) and the calcium-dependent K⁺ channelsexhibit six hydrophobic transmembranal domains, one of which (S4)contains repeated positive charges which confer on these channels theirsensitivity to voltage and, consequently, in their functional outwardrectification (Logothetis, D. E. et al., 1992, Neuron, 8, 531–540;Bezanilla, F. and Stefani, E., 1994, Annu. Rev. Biophys. Biomol.Struct., 23, 819–846).

The K⁺ channels with inward rectification (Kir families) have only twotransmembranal domains. They do not have the S4 segment and the inwardrectification results from a voltage-dependent blockade by cytoplasmicmagnesium (Matsuda, H., 1991, Annu. Rev. Physiol., 53, 289–298; Lu, Z.and Mackinnon, R., 1994, Nature, 371, 243–246; Nichols, C. G. et al.,1994, J. Physiol. London, 476, 399–409).

A common structural unit, called the P domain, is found in both groups,and constitutes an essential element of the structure of theK⁺-permeable pore. The presence of this unit in a membrane protein isconsidered to be the signature of the structure of a K⁺ channel (Pongs,O., 1993, J. Membrane Biol., 136, 1–8; Heginbotham, L. et al., 1994,Biophys. J., 66, 1061–1067; Mackinnon, R., 1995, Neuron, 14, 889–892;Pascual, J. M. et al., 1995, Neuron, 14, 1055–1063).

The present invention is based on the cloning of a K⁺ channel which isthe first member of a new structural and functional group of potassiumchannels. This new K⁺ channel has a novel molecular architecture withfour transmembranal segments and two P domains. From a functional pointof view, this channel is remarkable in that it exhibits weak inwardrectification properties. This new channel is referred to below asTWIK-1 following the English-language term “Tandem of P domains in aWeak Inward rectifying K⁺ channel”. Its abundance and its presence in alarge number of tissues are such as to confer on it a fundamental rolein the transport of potassium in a large number of types of cells.

The discovery of this new family of potassium channels and the cloningof a member of this family provides, notably, new means for screeningdrugs capable of modulating the activity of these new potassium channelsand thus of preventing or treating the diseases in which these channelsare involved.

The research activities that led to the cloning of the TWIK-1 channelwere carried out in the manner described below with reference to theattached sequences and drawings in which:

SEQ ID NO: 1 represents the nucleotide sequence of the cDNA of TWIK-1and the amino acid sequences of the coding sequence.

SEQ ID NO: 2 represents the amino acid sequence of the TWIK-1 protein.

FIGS. 1A–1D represents the Northern blot analysis, the nucleotidesequences and the deduced amino acid sequence, as well as thehydrophobicity profile and a schematic of TWIK-1. (A): expression ofTWIK-1 mRNA in human tissues; each track contains 5 μg of poly(A)⁺; theautoradiograph was exposed for 24 hours. (B): SEQ ID NO: 1. cDNAsequence of TWIK-1 and the amino acid sequences of the coding sequence.The supposed transmembranal segments are circled and the P domains areunderlined; o represents a potential glycosylation site and ▪ representsthe threonine residue in the consensus recognition site of proteinkinase C. (C): the hydrophobicity analysis and the topology of TWIK-1deduced from it; the hydrophobicity values were calculated according tothe method of Kyte and Doolittle (window size of 11 amino acids) and arepresented in relation to the position of the amino acid; the shadedhydrophobic peaks correspond to the transmembranal segments. (D): aschematic of TWIK-1, showing the configuration of the P1, P2 and M1-M4domains.

FIGS. 2A–2B represents the sequence alignments. (A): Highlighted portionof SEQ ID NO: 2 from FIG. 2B. alignment of the P domains of TWIK-1,TOC/YORK and other representative K⁺ channel families; the identical andconserved residues are circled in black and in gray, respectively. (B):SEQ ID NO: 2. alignment of TWIK-1 with potential homologues of C.elegans; the sequences M 110.2 and F17C8.5 were deduced from the genesequences (respective access numbers Z49968 and Z35719); thecomputerized splicing of the other genomic sequences of C. elegans(respective access numbers Z49889, P34411 and Z22180) is notsufficiently precise to allow their perfect alignment and is thereforenot shown.

FIGS. 3 a–3 f shows the biophysical and pharmacological properties of K⁺currents recorded by the imposed voltage technique on Xenope oocytesthat had received an injection of TWIK-1 cRNA; (a): the oocyte wasmaintained at a holding potential (HP) of −80 mV and the currents wererecorded at the end of 1-s voltage jumps from −120 to +60 mV in 20 mVincrements. (b): regular current-voltage relationship using the sametechnique as in (a). (c): potential reversal of the TWIK-1 currents(Erev) as a function of the external K⁺ concentration. (d): currenttracings linked to +30 mV depolarizations starting at a holdingpotential (HP) of −80 mV in the absence (top tracing) and in thepresence (bottom tracing) of 1 mM of Ba²⁺. (e): blocking effect of 100μM of quinine, same protocol as in (d). (f): dose-response relationshipof the blocking of the TWIK-1 currents by quinine.

FIGS. 4 a–4 c show the influence of the expression of TWIK-1 on themembrane potential. (a): dose-response relationships of the cRNA; toprow=equilibrium state of the outward currents measured at +30 mV; bottomrow=membrane potentials associated with the resting state. (b): effectof 100 μM of quinine on the membrane potential of an oocyte which didnot receive an injection (left tracing) and that of an oocyte thatreceived 20 ng of TWIK-1 cRNA. (c): statistical evaluation of thedepolarizing effects of 100 μM of quinine on oocytes that did notreceive injections (left bars) and on oocytes that received injectionsof 20 ng of TWIK-1 cRNA (right bars); control (unfilled bar), +quinine(solid bars); each bar represents the mean±SD of 5 oocytes.

FIGS. 5 a–5 c shows the properties of the single TWIK-1 channel. (a):current tracings recording in the input-output configuration to themembrane potentials indicated in the absence (m) or in the presence (.)of internal M²⁺ (3 mM) and in symmetry with 140 mM of K⁺. (b): mean ofcurves I–V (n=10). (c and d): open time of distribution obtained at +80mV (top histograms) and at −80 mV (bottom histograms) in the presence of3 mM Mg²⁺ (c) or in the absence of Mg²⁺ (d).

FIGS. 6 a–6 g shows the blocking of the TWIK-1 channels by the internalpH. (a and b): blocking effect of the internal acidification on theTWIK-1 currents, induced by perfusion of CO₂; (a): tracings ofsuperimposed currents induced by a depolarization phase at −30 mVstarting at HP=−80 mV, control (top tracing), effect when equilibrium isreached in the presence of CO₂ (bottom tracing); (b): graph (n=5)showing the almost complete blockade of the TWIK-1 currents induced byCO₂; (c and d): internal acidification induced by the application of DNP(1 mM). (c): same protocol as in (a), control (top tracing) and after 5minutes of application of DNP (bottom tracing); (d): graph (n=4)indicating the percentage of TWIK-1 current remaining after treatmentwith DNP. (e and f): imposed voltage (method: attached patch) undersymmetrical conditions of K⁺ concentration (140 mM) maintained at +80mV. (e): course over time of the effect of 1 mM of DNP (marked witharrow) on the activities of the single TWIK-1 channel. (f): graph (n=4)showing the effect of DNP on the mean probability of opening NP_(o)calculated during 1 minute of recording starting at the equilibriumstate. (g): activities measured in the “inside-out-patch” state at 80 mVat different internal pH values. Bar graph (n=10) of NP_(o) in relationto the internal pH.

FIGS. 7 a–7 d shows the activation of the TWIK-1 channels by PMA,activator of protein kinase C. (a): perfusion of PMA (30 nM) for 10minutes increases the TWIK-1 current (top tracing) induced by adepolarization phase at +30 mV starting at HP=−80 mV, control current(top tracing). (b): graph (n=5) showing the activation effect of PMA onthe TWIK-1 currents. (c and d): attached patch configuration undersymmetrical K⁺ concentration conditions maintained at +60 mV; (c):course over time of the effect of 30 nM of PMA on the single channelactivities; the recordings of the channel activity were performed with arapid scanning before and after the application of PMA; (d): bar graph(n=5) showing the activation effect of PMA on NP_(o).

The P domains of K⁺ channels were used to determine the correspondingsequences in the GenBank data base by means of the BLAST sequencealignment program (Altschul, S. F. et al., 1990, J. Mol. Biol., 215,403–410). There was thus identified a 298 pb human Tag expressedsequence (EST, HSC3AH031), the deduced amino acid sequence of whichincludes a nonconventional “P-like” domain sequence: GLG in place of GYGas shown in FIG. 2 a. It was then envisaged that this EST sequence was apartial copy of a mRNA coding a new type of K⁺ channel subunit. A DNAprobe was prepared from this sequence in order to carry outhybridization with a Northern blot (Clontech) of multiple human tissues.A 1.9 kb transcript was thereby found in abundance, as shown in FIG. 1a, in the heart and the brain and, at lower levels, in the placenta, thelung, the liver and the kidney. The DNA probe was used to screen a bankof kidney cDNA and four independent clones were obtained. The cDNAinserts of 1.8 to 1.9 kb of these clones all have the same open readingframe (ORF) containing a regio identical to the 298 pb sequence ofHSC3AH031 and differing solely in the length of their noncoding 5′sequences.

Primary Structure of TWIK-1

The following characteristics were demonstrated:

The Sequences of the cDNA clones contain an ORF of 1011 nucleotidescoding for a polypeptide of 336 amino acids shown in FIG. 1 b.

This protein has two P domains.

Other than the P domains, no significant alignment was seen betweenTWIK-1 and K⁺ channel recently cloned in yeast and which also has two Pdomains (Ketchum, K. A., et al., 1995, Nature, 376, 690–695).

Analysis of the hydrophobicity of TWIK-1, shown in FIG. 1 c, reveals thepresence of four transmembranal domains, designated M1 to M4.

By placing the NH2 end on the cytoplasmic surface, in accordance withthe absence of signal peptide, one obtains the topology model shown inFIG. 1 c.

In this model, the two P domains are inserted in the membrane from theexterior in accordance with the known orientation of these loops in theK⁺ channels.

In addition, the general structural unit of TWIK-1 is similar to theunit that one would obtain by making a tandem of two classical subunitsrectifying the entry of a potassium channel. Like a classical inwardrectifier, TWIK-1 does not exhibit the highly conserved segment S4 whichis responsible for the sensitivity to the membrane potential of theinward rectification of the K⁺ channels of the Kv family.

A nonusual large loop of 59 amino acids is present between M1 and P1,such as to extend the length of the linker M1-P1 of the extracellularside of the membrane.

A potential site of the N-glycosylation is present in this loop.

Three consensus sites of phoshporylation are present at the N-terminal(Ser 19 for calcium calmodulin kinase II) and C-terminal (Ser 303 forcasein kinase II) ends of the cytoplasmic domains, and in the M2-M3linker (Thr161 for protein kinaseII).

The alignment of the P domains of an important group of K⁺ channels ispresented in FIG. 2 a. It shows that the regions constituting the poreselective for K⁺ are well conserved including the G residues in position16 and 18 and three other residues indicating practically exclusivelyconservative change in positions 7, 14 and 17. It is of interest to notethat a leucine residue is present in the place of a tyrosine conservedin position 18 in the P2 domain of TWIK-1, or of a phenylalanine inposition of 17 of the P domain of the K⁺ channel of type eag.

The Homologues of TWIK-1

Comparison of the complete sequence of TWIK-1 with the sequences of theGenbank data base allowed identification of at least five genes ofCaenorhabditis elegans which had been characterized in the context ofthe Nematode Sequencing project, and which potentially code forstructural homologues of TWIK-1. The alignment of two of thesehomologues with TWIK-1 is shown in FIG. 2 b. The homologies of totalsequences between the deduced proteins of C. elegans and TWIK-1 arecirca 55 to 60% and circa 25 to 28% of identity. The homologies amongsequences of C. elegans are not higher.

Functional Expression of TWIK-1

For the functional study, the coding sequence of TWIK-1 was insertedbetween the noncoding sequences 5′ and 3′ of Xenopus globin in thevector pEXO (Lingueglia, E. et al., 1993, J. Biol. Chem., 269,13736–13739). A complementary RNA (cRNA) was transcribed of thisconstruction and injected in the oocytes of X. laevis. A noninactivatingcurrent, free from noninjected cells, was measured by the imposedvoltage technique, as shown in FIG. 3 a. Kinetic activation of thecurrent is usually instantaneous and cannot be resolved because it ismasked by the capacitive discharge of the current recorded at thebeginning of the impulse. The current-voltage relationship is linearabove 0 mV and then saturates for a stronger depolarization of themembrane, as shown in FIG. 3 b. TWIK-1 is therefore K⁺ selective. In thecase of a replacement of the external K⁺ by Na⁺ or N-methyl-D-gluconate,the reversal of the potential of the currents follows the K⁺ equilibriumpotential (E_(K)), as shown in FIG. 3 c. In addition, a change by 10 inthe concentration [(K)]_(o) leads to a change of 56±2 mV in theinversion value of the potential, in accordance with Nernst's equation.

As shown in FIG. 3, the K⁺ currents of TWIK-1 are inhibited by Ba²⁺(FIG. 3 d) with an IC₅₀ value of 100 μM, by quinine (FIGS. 3 e and 3 f)and by quinidine (not shown) with respective IC₅₀ values of 50 and 95μM. The TWIK-1 currents are slightly sensitive to TEA and to the classIII antiarrhythmic agent tedisamil (30% inhibition for each, at 20 mMand 100 μM, respectively). Less than 10% inhibition was seen afterapplication of 4-aminopyridine (1 mM), apamin (0.3 μM), charybdotoxine(3 nM), dedrotoxine (0.1 μM), clofilium (30 μM), amiodarone (100 μM) andglibenclamide (30 μM). The TWIK-1 channel is not sensitive to the K⁺channel openers cromakaline (100 μM) and pinacidil (100 μM).

FIG. 4 shows the effect of increasing the doses of injected TWIK-1 cRNAon the independent expression of the time of the K⁺ currents and on theresting state of the membrane potential (E_(m)). As soon as the currentappears, the oocytes become increasingly polarized, reaching a value ofE_(m) close to E_(K). The amplitude of the TWIK-1 current reaches valuesof 0.6 to 0.8 μM with the injection of 20 ng per oocyte. Higher doses ofTWIK-1 cRNA are toxic, leading to a reduction in expression. In oocytesthat received 20 ng of cRNA, quinine is the best blocker of TWIK-1,inducing a noteworthy reversible depolarization (73±6 mV, n=5) as shownin FIGS. 4 b and 4 c.

The Unitary Properties of the TWIK-1 Channel

Single channel current recordings, shown in FIG. 5, in an inside-outpatch configuration or in a whole cell configuration show that theTWIK-1 channels assure the passage of influx or exit currents as afunction, respectively, of a depolarization or a hyperpolarization (FIG.5 a). The current-voltage relationship of the single channel, shown inFIG. 5 b, shows a barely accentuated inward rectification in thepresence of 3 mM (FIG. 5) and 10 mM (not shown) of Mg²⁺ on thecytoplasmic side. As shown in FIG. 5 b, this rectification disappears inthe absence of internal M²⁺. With 3 mM of internal Mg²+, the meanduration of opening at +80 mV is 1.9 ms and the unitary conductance is19±1 pS (FIG. 5 c). At −80 mV, the channels are oscillating with a meanduration of opening of 0.3 ms, and a conductance value increasing to34±pS. The withdrawal of the internal Mg²⁺ ions does not influence thekinetic parameters under either polarized or depolarized conditions, butthe unitary conductance measured at −80 mV reaches 35±4 pS. Thisapparent increase in conductance in the single channel suggests that itis the extremely rapid oscillation induced by Mg²⁺ that results in anunderestimation of the real value of conductance. The same propertieswere observed in the fixed cell configuration, showing that the channelbehavior is not modified by the excision of the patch. The TWIK-1channels in the excised patches do not discharge and do not appear to bedeficient in intracellular constituents. In contrast to numerouschannels which require the presence of ATP for their activity in theexcised patch configuration, ATP is not required for the expression ofTWIK-1. In addition, perfusion of the patch with a solution containing10 mM of ATP does not induce any effect on the activity of the TWIK-1channel.

The Activity Regulation Properties of the TWIK-1 Channel.

The intracellular pH (Ph_(i)) is involved in the control of numerouscellular processes, and in cells such as the hepatic cells, the changein Ph_(i) regulates the membrane potential (Bear, C. E. et al., 1988,Biochim. Biophys. Acta, 944,113–120).

Intracellular acidification of the oocytes was produced using twomethods:

superfusion with a solution enriched in CO₂ which produces acidificationby a mechanism involving the bicarbonate transport system (Guillemare,E. et al., 1995, Mol. Pharmacol., 47, 588–594);

treatment with dinitrophenol (DNP), which is a metabolic inhibitor thatdecouples the H⁺ gradient in mitochondria and induces internal acidity(Pedersen, P. L. and Carafoli, E., 1987, Trends Biol. Sci., 12,146–189).

Both of these experimental methods resulted in a significant reductionin the TWIK-1 currents, greater than 95% in the case of CO₂ and 80% inthe case of DNP of the control amplitude values, as shown in FIGS. 6 ato 6 d. The inhibition induced by DNP on the activity of the single K⁺channel was again observed under the attached patch conditions, as shownin FIGS. 6 e to 6 f. However, after excision of the patch, the activityof the channel became insensitive to the acidification of the internalsolution produced either by modifying the Na₂HPO₄/NaH₂PO₄ buffer ratio(FIGS. 6 g and 6 h) or by bubbling of CO₂ (not shown). Thus, the effectof the pH value on the activity of the TWIK-1 channel is probablyindirect.

Phosphorylation or dephosphorylation of specific amino acid residues isan important mechanism of regulation of the ionic channels (Levitan, I.B., 1994, Annu. Rev. Physiol., 56, 193–212). As shown in FIG. 7,activation of protein kinase C by phorbol-12 myristate acetate (PMA, 30nM) increases the TWIK-1 currents. The inactive phorbol ester4α-phorbol-12, 13 didecanoate (PDA, 1 μM) has no effect. In an attachedpatch which initially expressed solely a single channel, application ofPMA . . . the presence of at least five channels (FIGS. 7 c and 7 d).This experiment shows that at least four channels are silently presentin the patch before the application of PMA. Since the TWIK-1 sequencecontains a consensus phosphorylation site for protein kinase C (PKC),located at the level of the threonine in position 161 (FIG. 1 b), theeffect of PMA suggests regulation under the control of PKC. However, themutation of the threonine 161 into alanine leads to a muted channelwhich remains functional and conserves the capacity to be activated byPMA.

Activation of protein kinase A by application of 8-Cl-AMPc (300 μM) orforskolin (10 μM) does not affect the activity of TWIK-1. Elevation ofthe cytoplasmic Ca²⁺ concentration by application of A23187 (1 μM) whichcould be activated by Ca²⁺-calmodulin kinase II and/or reveal thepresence of a channel activated by the Ca²⁺, is also without effect onthe properties of the TWIK-1 channel.

Thus, the object of the present invention is an isolated, purifiednucleic acid molecule that codes for a protein constituting a TWIK-1potassium channel or exhibiting the properties and structure of the typeof the TWIK-1 channel described above.

More specifically, the said nucleic acid molecule codes for the TWIK-1protein, the amino acid sequence of which is represented in the attachedsequence list as number SEQ ID NO: 2, or a functionally equivalentderivative of this sequence. Such derivatives can be obtained bymodifying and or suppressing one or more amino acid residues of thissequence, as long as this modification and/or suppression does notmodify the functional properties of the TWIK-1 potassium channel of theresultant protein.

The sequence of a DNA molecule coding for this protein is morespecifically the molecule coding for TWIK-1 represented in the attachedsequence list as number SEQ ID NO: 1.

The invention also relates to a vector containing a molecule of theaforementioned nucleic acid, as well as a procedure for production orexpression in a cellular host of a protein constituting a TWIK-1potassium channel or a channel of the same family as TWIK-1.

A procedure for production of a protein constituting a TWIK-1 potassiumchannel or exhibiting the properties and structure of the type of theTWIK-1 channel consists of:

transferring a nucleic acid molecule of the invention or a vectorcontaining the said molecule into a cellular host,

culturing the cellular host obtained in the preceding step underconditions enabling the production of potassium channels exhibiting theproperties of TWIK-1,

isolating by any suitable method the proteins constituting the potassiumchannels of the TWIK-1 family.

A procedure for expression of a TWIK-1 potassium channel or a potassiumchannel of the same family as TWIK-1 consist of:

transferring a nucleic acid molecule of the invention or a vectorcontaining the said molecule into a cellular host,

culturing the cellular host obtained in the preceding step underconditions enabling the expression of potassium channels of the TWIK-1family.

The cellular host employed in the preceding procedures can be selectedfrom among the prokaryotes or the eukaryotes, and notably from among thebacteria, the yeasts, mammal cells, plant cells or insect cells.

The vector used is selected in relation to the host into which it willbe transferred; it can be any vector such as a plasmid.

The invention thus also relates to the transferred cells expressing thepotassium channels exhibiting the properties and structure of the typeof the TWIK-1 channel obtained in accordance with the precedingprocedures.

The cells expressing TWIK-1 potassium channels or channels exhibitingthe properties and structure of the type of the TWIK-1 channels obtainedin accordance with the preceding procedures are useful for the screeningof substances capable of modulating the activity of the TWIK-1 potassiumchannels. This screening is carried out by bringing into contactvariable amounts of a substance to be tested with cells expressing theTWIK-1 channel or potassium channels exhibiting the properties andstructure of the type of the TWIK-1 channels, then measuring, by anysuitable means, the possible effects of said substance on the currentsof the potassium channels of these channels.

This screening procedure makes it possible to identify drugs that usefulin the treatment of diseases of the heart or of the nervous system.Diseases involving the potassium channels and thus likely to involve thechannels of the TWIK-1 family are, for example, epilepsy, heart(arrhythmias) and vascular diseases, neurodegenerative diseases,especially those associated with ischemia or anoxia, the endocrinediseases associated with anomalies of hormone secretion, musclediseases.

An isolated, purified nucleic acid molecule coding for a proteinconstituting a TWIK-1 potassium channel or a vector including thisnucleic acid molecule or a cell expressing the TWIK-1 potassiumchannels, are also useful for the preparation of transgenetic animals.These can be animals supra-expressing the said channels, but especiallyso-called knock-out animals, i.e., animals presenting a deficiency ofthese channels; these transgenetic animals are prepared by methods knownto the experts in the field, and enable the preparation of live modelsfor studying animal diseases associated with the TWIK-1 channels.

The nucleic acid molecules of the invention or the cells transformed bysaid molecule can also be used in genetic therapy strategies forcompensating for a deficiency in the potassium channels at the level ofone or more tissues of a patient. The invention thus also relates to amedication containing nucleic acid molecules of the invention or cellstransformed by said molecule for the treatment of disease involving thepotassium channels.

In addition, the gene of the TWIK-1 channel has been located onchromosome 1 at position q42-q43. The chromosomal localization of thisgene constitutes a determinant result for the identification of geneticdiseases associated with this new family of potassium channels; thus,the knowledge of the structure of the TWIK-1 family of channels is suchas to allow performance of a prenatal diagnosis of such diseases.

The present invention also has as its object a new family of K⁺channels, of which TWIK-1 is a member, which are present in most humantissues and especially abundant in the brain and the heart, and whichexhibit the properties and structure of the type of those of the TWIK-1channels described above. Thus it relates to an isolated, purifiedprotein whose amino acid sequence is represented in the attachedsequence list as number SEQ ID NO: 2, or a functionally equivalentderivative of this sequence.

Such derivatives can be obtained by modifying and/or suppressing one ormore amino acid residues of this sequence or by segmenting thissequence, as long as this modification and/or suppression or deletion ofa fragment does not modify the functional properties of the TWIK-1 typepotassium channel of the resultant protein.

A protein constituting a TWIK-1 type potassium channel is useful for themanufacture of medications intended for the treatment or prevention ofdiseases involving dysfunction of the potassium channels.

Polyclonal or monoclonal antibodies directed against a proteinconstituting a TWIK-1 type potassium channel can be prepared by theconventional methods described in the literature.

These antibodies are useful for investigating the presence of potassiumchannels of the TWIK-1 family in different human or animal tissues, butthey can also find applications in the therapeutic domain, due to theirspecificity, for the in vivo inhibition or activation of TWIK-1 typepotassium channels.

Other advantages and characteristics of the invention will be madeobvious from the examples below which are nonlimitative examples relatedto the cloning and expression of TWIK-1.

Identification of the HSC3AH031 EST Sequence and Analysis of the RNA

The P domains of the cloned channels were used to investigate homologuesin the NCBI (National Center of Biotechnology) data bases using thesequence alignment program tBLASTn. Translation of an EST sequence(HSC3AH031, Genbank access number: F12504) presented a significantsequence similarity (P=1.2×10⁻³) with the second P domain of a yeast K⁺channel. This 298 pb sequence was originally obtained from a human braincDNA bank in the context of the Genexpress cDNA program (Auffray, C. etal., 1995, C. R. Acad. Sci., III, Sci. Vie, 318, 263–272). A 255 pb DNAfragment corresponding to HSC3AH031 was amplified by PCR from cDNAderived from human brain poly(A)⁺ and subcloned in pBluescript(Stratagene) to yield pBS-HSC3A.

For the RNA analysis, a Northern blot of multiple human tissues(Clontech) was screened with the pBS-HSCA insert tagged with P³² in 50%formamide, 5× SSPE (0.9 M NaCl; 50 mM sodium phosphate; pH 7.4; 5 m MEDTA), 0.1% SDS, 5× Denhardts, 20 mM potassium phosphate, pH 6.5 and 250μg of salmon sperm DNA denatured at 55° C. for 18 hours. The blots werewashed to a final stringency of 0.1 SSC (3 M NaCl; 0.3 M sodium citrate;pH 7.0), 0.3% SDS at 65° C.

Isolation of the cDNA Cloning TWIK-1

An oligo(dT) cDNA bank stemming from poly(A)⁺ RNA isolated from humanadult kidney was screened with the pBS-HSCA insert tagged with p³². Thefilters were hybridized in 50% formamide, 5×SSC, 4× Denhardt, 0.1% SDSand 100 μg of salmon sperm DNA denatured at 50° C. for 18 hours. Fourpositive hybridization clones were isolated from circa 5×10⁵ clones. TheλZAPII phages containing the cDNA inserts were converted into cDNAplasmids (Stratagene). The DNA inserts were characterized by restrictionenzyme analysis and by total or partial DNA sequencing on both strandsusing the dideoxy nucleotide method on an automated sequencer (AppliedBiosystems 373A).

Mutations, cRNA Synthesis and Oocyte Injection.

The TWIK-1 coding sequence was amplified using a low-error rate DNApolymerase (Pwo DNA pol, Boehringer) and subcloned in the plasmid pEXOiiso as to yield pEXO-TWIK-1. Mutations were performed using the wholeplasmid pEXO-TWIK-1 with a highly reliable PCR extension kit(Boehringer) and two adjacent primers. One of these introduced apunctiform mutation in the TWIK-1 coding sequence, changing the 161 Thrcodon into a codon for alanine. The product of the PCR was linearized bythe enzyme BamHI and the cRNA were synthesized using a T7 RNA polymerase(Stratagene). Preparation of the X. laevis oocytes and cRNA injectionwere carried out in accordance with the literature (Guillemare, E. etal., 1992, Biochemistry, 31, 12463–12468.

Electrophysiological Measurements.

In a 0.3-ml perfusion chamber, a single oocyte was impaled on twostandard glass microelectrodes (0.5–2.0 MW) charged with 3 M KCl andmaintained under voltage-clamp with a Dagan TEV200 amplifier. The bathsolution contained 98 mM KCl, 1.8 mM CaCl₂, 2 mM MgCl₂ and 5 mM HEPES atpH 7.4 with KOH. Stimulation of the preparation, data acquisition andanalyses were carried out with the pClamp program (Axon Instruments,USA).

For the patch-clamp experiments, the vitelline membrane was removed fromthe oocytes as described in the literature (Duprat, F. et al., 1995,Biochem. Biophys. Res. Commun., 212, 657–663); the oocytes were thenplaced in a bath solution containing 140 mM KCl, 1.8 mM CaCl₂, 2 mMMgCl₂ and 5 m M HEPES at pH 7.4 with KOH. The pipettes were filled witha strong K⁺ solution (40 mM KCl, 100 mM of potassium methane sulfonate,1.8 mM CaCl₂, 2 m M MgCl₂ and 5 mM HEPES adjusted to pH 7.4 with KOH).100 μM of GdCl₃ was added to the pipette solution to inhibit the actionof the activated channels. The inside-out patches were perfused with asolution containing 140 m M KCl, 10 mM CaCl₂, 5 mM HEPES adjusted to pH7.2 with KOH and 5 mM EGTA added daily. The single channel signals werefiltered at 3.5 kHz and analyzed with the Biopatch program (Bio-Logic,Grenoble, France).

1. An isolated and purified tandem of P domains in a weak inwardrectifying potassium channel (TWIK-1) protein constituting a potassiumchannel, wherein the protein comprises SEQ ID No. 2.